VIROLOGY

89,

547-559

Purification Glycoprotein

(1978)

and Characterization of a Murine of 75,000 Daltons that is Related Glycoprotein of Murine Leukemia W. L. McLELLAN

Clnnwr

Riology

Pmgrom,

NCI

Frederick

AND

Cancer

Accepted

J. N. IHLE

Kesmrch

May

Tumor Cell Surface to the Major Envelope Virus

Cenfrr,

Frederick,

Mar.vlnnd

21701

31, 1978

An antigen which competes with murine C-type viral glycoprotein in an interspecies radiolmmunocompetition assay has been purified from the surface of EL-4 tumor cells. The EL-4 tumor cell is virus particle negative. The amount of p30 was extremely low and there was no detectable p12 or ~15, confirming that the tumor cell was not expressing a murine oncornavirus. In homologous competition radioimmunoassays, the tumor cell was negative for Rauscher and AKR gp71, but in an interspecies assay employing IL”I-Rauscher gp71 and anti-feline leukemia virus serum, there was a reactive antigen. The gp’il-like antigen was on the surface of the tumor cell since, by immunofluorescence and immunoelectronmicroscopy, the EL-4 cell could be labeled with antiserum against Rauscher virus gp71. The gp71 cross-reactive antigen was purified by lithium diiodosalycilate extraction, DEAF: chromatography, and lentil lectin affinity chromatography. It is a glycoprotein of about 75,000 daltons which could be precipitated by various broadly reactive antisera to murine gp7ls and antisera to murine virus. Based on radioimmunocompetition assays, the purified gp75 was not, related to AKR or Rauscher gp71, but did compete in an assay using BALB/2 xenotropic gpil and anti-C57/L virus serum. It also competed in an assay Iusing ‘“‘IMoloney virus gp71 and anti-Moloney virus serum, but not in a more type-specific assay using ‘%Moloney virus gp71 and anti-Molonry gpil serum. l’ryptic peptidr maps of iodinated proteins were prepared and the cell surface antigen was compared with AKR gp71, Maloney gp71, Rauscher gp71, and xenotropic BALB/B and NZB gp’ils. The EL-4 gpT5 was not identical with or very similar structurally to any of the viral gp7ls. The differences in the structures of the various viral gp71s shown here and also in other laboratories are consistent with the idea that the em genes of murine viruses which code for gp7ls are sites of frequent recombinational events. Recombination between different endogenous viral sequences or between viral and host allelic genomes could have resulted in many immunologically related proteins on viruses, cell surfaces and, in sera of mice, which are immunologically relatrd, about 70,OOWdalton molecular weight, hut have divergent structure. INTRODUCTION

There has been an increasing awareness of the serological diversity of the major envelope glycoprotein of murine leukemia viruses (MuLV) since Ihe purification of this 71,000-dalton glycoprotein from Rauscher virus (Strand and August, 1973), Friend virus (Moennig et ul., 1974), AKR virus (Ihle et al., 1976a), Moloney virus (Kennel, 1976)) Scripps virus (Kennel, 1976), and xenotropic isolates (McClintock et al., 1977) and the development of various

antisera. Serological data toget,her with some data on structural diversity are presently forming one basis for murine C-type viral subclassification (Elder et nl., 1977a). The major envelope glycoprotein of MuLV (gp71), as well as molecules closely related to it, can be expressed on the surfaces of mouse cells (Kennel and Feldman, 1976). There can be expression of a viral gene (e.g., gp71) on the cell surface without expression of complete virus (Hino et al., 1976; Kennel and Feldman, 1976; Strand et al., 1974). A 70,000-dalton glycoprotein 547 0042-6822/78/0892-0.547$02.00/O Copyright 0 1978 by Academic Press, Inc All rights of reproduction in any form reserved.

548

McLELLAN

which cross-reacts antigenically with viral gp71 has been found on the surface of lymphoblasts that produce virus as well as on the surface of thymocytes in the absence of detectable virus production (Lerner et al., 1975). Distinct gp7ls have been found free in the serum of mice and on sperm and in seminal fluid (Del Villano and Lerner, 1976; Elder et al., 1977a); an AKR type gp71 has been found in t,he bone marrow of C57BL/6 mice (McClintock et al., 1977). Differentiation alloantigens of the mouse have been shown to be serologically related to MuLV gp71. Gix, a serologically characterized cell surface antigen, is found on thymocytes of strains of mice (e.g., 129), which are not overt virus producers (Stockert et al., 1971, 1975). It is normally absent from other strains but appears on the surface of lymphocytes of these genotypically Gix- mice with the occurrence of leukemia (Stockert et al., 1971). Gix antigen was reported to be the cell surface expression of the major glycoprotein of MuLV (Obata et al., 1975), but more recent data have suggested that it is expression of an allele or is a subset of the viral glycoprotein (Tung et al., 1975a). Structural variants of the molecule have been proposed: “0 gp70” in the C57BL/6 strain (Tung et al., 1975a), and “X gp70” in RAD-Al or ASLl leukemia (Tung et al., 1976). These variants can be precipitated from surface-labeled cells by the antiserum defining Gix (anti-NTD serum), but thymocytes of these strains do not absorb the cytotoxic activity of the antiserum toward 129 thymocytes. An increasing list of antigenically related 70,000dalton glycoproteins has led to the speculation that the gp7ls are polymorphic products of a large multigene family including viral and differentiation antigens (Elder et al., 1977a). To elucidate the relatedness and probe the significance of the diverse cell surface gp7ls from various strains of mice, the glycoproteins have to be isolated and characterized biochemically as well as antigenitally. The ideal sources are tumors or tissues that are not overtly producing virus. A protein cross-reactive with Rauscher MuLV gp71 in an interspecies assay has been isolated from ascites fluid of NZB

AND

IHLE

mice using immunoaffmity chromatography (Kennel, 1976). By tryptic peptide mapping, this 70,000-dalton glycoprotein has been found to differ from the glycoprotein of Friend, Moloney, Rauscher, and Scripps murine C-type viruses. Antigenitally, it differed from the ecotropic AKR virus, the FMR group of viruses, and Scripps virus, but was to some extent related to xenotropic virus. However, the NZB strain of mouse is neither GIx- nor a nonvirus producer (Levy, 1973), so the isolated glycoprotein could be of viral origin. In the present study, a glycoprotein which reacts in an interspecies gp71 assay has been isolated from the GixGSCA-(Aoki et al, 1977) EL-4 tumor cell, a chemically induced (Gorer, 1950) virusnegative tumor of C57BL/6 mice which is passaged in ascites form. The glycoprotein has been examined serologically and structurally and found to be distinct from any of the viral gp7Os examined. Serologically, it was most closely related to xenotropic virus gp71, but by tryptic peptide fingerprinting it was quite different. MATERIALS

AND

METHODS

Tumor cells. The EL-4 tumor line is from a stock known to be free of pathogenic murine viruses. Approximately 2 x 10’ cells were injected intraperitoneally into 8 to loweek-old C57BL/6 mice and 8 days later the ascitic fluid was aspirated. Cells were harvested by centrifugation for 5 min at 500 g, and the red cells were lysed by adding 4 volumes of 0.83% NH&l in 0.01 it4 Tris-HCl buffer, pH 7.5, at 4’. The cells were harvested by centrifugation, washed and suspended in minimal essential medium (MEM). The yield was approximately 8 X 10’ cells per mouse and the viability was greater than 95% judged microscopically by trypan blue exclusion (Boyse et al., 1964). ESG2 tumor cells were obtained by teasing apart spleens of C57BL/6 mice injected with 2 x lo7 E$G2 cells 8 days before killing. AKR thymomas were spontaneous tumors appearing in 6 to g-month-old AKR mice. Red cells were lysed and tumor cells were washed as above, except AKR thymomas were suspended in RPM1 1640 medium. Antisera. Goat anti-Rauscher gp71 was

CELL

SURFACE

ANTIGEN

obtained from Dr. L. Charmella, Frederick Cancer Research Center (FCRC); rabbit anti-AKR gp71 and rabbit anti-Friend gp71 and rabbit anti-Moloney gp71 were prepared in our laboratory. Goat anti-feline leukemia virus and goat anti-Moloney virus sera were provided by Dr. R. Wilsnack (Huntington Research Center, Brooklandville, Md.) and goat anti-C57/L virus IgG by Dr. R. Gilden (FCRC). Rabbit anti-goat gamma globulin and goat anti-rabbit gamma globulin were prepared in this laboratory or purchased from Cappel Laboratories (Downingtown, Pa.). Assay for cross-reactive gp71. An interspecies competition radioimmunoassay employing anti-feline leukemia viruq (FeLV) serum and “)‘I-Rauscher gp71 was used (Strand and August, 1973). Approximately 5 ng ‘““I-Rauscher gp71 was incubated with dilutions of competing antigen, carrier goat gamma globulin and an amount of antiserum predetermined to give 50% of maximum precipitation of antigen. The total volume was 0.2 ml. The mixture was incubated for 3 hr at 37’; rabbit anti-goat gamma globulin was added and incubation continued at 4” for 18 hr. Precipitates were washed and counted. Standard competition curves were run with Rauscher gp71 to calculate the relative quantity of antigen. Competition assays were also performed with ““I-Rauscher gp71 and goat antiRauscher gp71 serum, lZ”I-AKR gp71 and rabbit anti-AKR gp71 serum (Ihle et al., 1976a), ‘“;‘I-BALB/2 gp71 and anti-C57/L virus serum (McClintock et al., 1977), and with ““I-Maloney MuLV gp71 and goat anti-Moloney MuLV serum. Competition assays were also performed for p30 and ~12 (Ihle et al., 1976b). When assays were performed on cells, the cells were first disrupted with 0.5% Nonidet P-40 (NP-40, Particles Data Laboratories, Elmhurst, Ill.) in phosphate-buffered saline at 37” for 15 min, and the extract was clarified by centrifugation at 15,000 g for 15 min. Viral antigens. The proteins were prepared as previously described (Ihle et al., 1976a; Ihle et al., 1976b; McClintock et al., 1977) and iodinated by the chloramine-T method of Hunter (1975). Purity of viral antigens was monitored by sodium dodecyl

RELATED

TO VIRAL

g~,;l

549

sulfate (SDS) gel electrophoresis, performed on slab gels with a 5-2070 gradient of acrylamide (Maizel, 1971). Proteins were stained with 0.25% Coomassie brilliant blue in 7% acetic acid, 40% methanol. Other methods. Labeling of glycoprotein with galactose oxidase and [“HIsodium borohydride and labeling of sialic acid by mild periodate oxidation and borotritide reduction has been described previously (McLellan and August, 1976). Tritium-labeled proteins were detected by fluorography of dried gels at -70” using Kodak RP 54 film (Bonner and Laskey, 1974). Lens culinaris hemagglutinin (LcH) -Sepharose was prepared by coupling the lectin to cyanogen bromide-activated Sepharose (Pharmacia Corp., Piscataway, N. J.) according to the procedure described by Hayman and Crumpton (1972). (LcH lectin was from Boehringer Mannheim, Indianapolis, Ind.). Protein was assayed by the method of Lowry et al. (1951) or a modification of tbis method (Wang and Smith, 1975) if lithium diiodosalycilate or interfering detergent was present. Tryptic peptide analysis. The iodinated sample was electrophoresed on a 5-20% SDS acrylamide slab gel and a radioautogram was made of the wet gel. The radioactive band was cut from the gel, ground finely, and extracted at 4” overnight with 0.1% SDS-O.05 M ammonium bicarbonate buffer, pH 8.0; after filtration to remove acrylamide, 200 pg bovine serum albumin was added and the sample was lyophilized. The sample was dissolved in 6 M urea 0.05 M NH,HCO:, and treated with 0.1 ml Dowex l-X2 (acetate form) to remove SDS (Weber and Kuter, 1971). Urea was removed by overnight dialysis against 0.02 M NHhHCO:s, pH 8.0; the sample was lyophilized and dissolved in 0.4 ml of the same buffer. Twenty-five micrograms TPCK trypsin (Worthington Biochemicals, Freehold, N. J.) were added and the sample was incubated at 37’ for 2 hr: an additional 25 pg TPCK trypsin were then added and incubation continued for 18 hr. The digest was applied to a 0.2 ml column of Dowex 50-X2 (H’ form); the column was washed with distilled water and radioactivity eluted with 2 N NH,OH. The eluate was lyophi-

550

McLELLAN

AND

IHLE

lized and the sample was dissolved in chrotemperature with 0.1 M o-methyl mannomatography solvent (1-butanol/pyridine/ side in PBS. Protein concentration was folacetic acid/water, 90:60:18:72), spotted on lowed by absorbance at 280 nm. The lectin a O.l-mm cellulose plate (20 X 20 cm, Cel affinity column was subsequently eluted 300-10 Machery-Nagel, Brinkmann). After with 1 M glycine buffer, pH 3.0, and the chromatography and thoroughly drying the eluted fractions were immediately neutralplates, electrophoresis was carried out at ized with 4 M Tris-base. The fractions right angles with pH 3.6 pyridine-acetate eluted with n-methyl mannoside were (0.375 1M) for 60 min at 1200 V (Oroszlan et pooled, concentrated by dialysis against dry al., 1974). Radioactive peptides were visuSephadex G-200 (Pharmacia), and then dialized by exposure of the plate to NS54T x- alyzed against 0.01 M Tris-HCl buffer, pH ray film (Kodak) or exposure at -70” to 8.0. The sample was rechromatographed on RP54 x-ray film using a Lightning Plus a 0.6 x 3-cm column of DEAE-cellulose Cronex intensifying screen (DuPont). with a linear gradient of NaCl O-O.3 M in Purification of cell surface glycoprotein. 0.01 M Tris-HCl, pH 8.0 (40 ml total volEL-4 cells were suspended in MEM meume) . dium; 0.06 M lithium diiodosalycilate (EastRESULTS man Organic Chemicals) was slowly added The EL-4 tumor cell does not express with stirring to a final concentration of 3 an ecotropic virus. Immunofluorescence mM, and PMSF (phenylmethylsulfonyl fluand immunoelectronmicroscopic studies oride, Sigma) was added to a final concentration of 2 mM. In a typical preparation, 3 showed that the EL-4 tumor could be lax 10”’ cells, obtained from ascites fluid of beled with antisera to Rauscher virus gp71 30 mice, were used. The cells were shaken but, not with antisera against AKR virus gently at room temperature for 30 min and gp71. Examination of the tumor cells by then separated by centrifugation at 700 g electronmicroscopy showed that there were for 10 min. The cells were re-extracted with no virus particles budding from cells (less 4 rnJ4 lithium diiodosalycilate (LDS) and 2 than one particle/300 cells) and that the antigen, being labeled with ferritin-conjumM PMSF. The extracts were centrifuged at 4” for 15 min at 15,000 g and solid am- gated antibody, was on the cell surface. To monium sulfate (42 g/100 ml) was added to quantitate and extend these observations, give a 70% saturated solution. The precipicompetition radioimmunoassays were pertate was allowed to form for 1 hr at 2” and formed with different viral antigens. Table 1 compares EL-4 cells with two virus-prothen collected by centrifugation, dissolved ESGS, a Gross virus-inin 0.01 M Tris-HCl buffer, pH 8.0, and ducing tumors, dialyzed against the same buffer. After cenduced, passaged, splenic tumor of C57BL/6 mice, and a spontaneous AKR thymoma. trifugation at 100,000 g for 1 hr, the superExtracts of EL-4 did not compete with nate was applied to a 1.5 x 40-cm column of DEAE-cellulose (DE52 Whatman) that TABLE 1 had been equilibrated with 0.01 M Tris-HCl COMPETITION RADIOIMMUNOASSAYOFEXTRACTSOF buffer, pH 8.0. The column was washed MURINE LEUKEMIA CELLS with 0.01 M Tris-HCl, pH 8.0, until no Nanograms/milligram of further protein eluted and then developed protein with 0.15 M NaCl, 0.01 M Tris-HCl, pH 8.0. EL-4 AKR-T EdG2 Fractions were collected and assayed for protein and for activity by competition raPP71 dioimmunoassay as described above. The AKR MuLV ND” 65 47 active fractions were applied to a 0.9 X 8Rauscher MuLV ND ND ND Inkrspecies 350 950 375 cm column of LcH-Sepharose equilibrated p30 48 1270 502 with phosphate-buffered saline (PBS) and p12 (AKR) ND 77 200 the column was washed with PBS at 4” ~15 (AKR) ND 100 16 until no further protein eluted. The lectin__-~ -__ ” ND, not detectable. Sepharose column was then eluted at room

CELL

SURFACE

ANTIGEN

AKR virus gp71 or Rauscher or Friend virus gp71 in homologous competition assays; however, in the interspecies assay employing ““I-Rauscher virus gp71 and antiFeLV serum, there was competition. The amount of p3O in extracts of EL-4 was extremely low compared with that in the E$G2 tumor or the AKR thymoma. In addition, there was no detectable p12 or pl5 in homologous competition assays with AKR p12 and ~15 nor was any Rauscher p12 or Moloney p15 detected. Therefore, we felt assured that the tumor cell was not producing a virus and what we were isolating was a cell surface protein. Purification of cell surface glycoprotein. The interspecies competition radioimmunoassay was used to follow the activity of the antigen being purified from the EL-4 tumor cell. Figure 1A shows competition curves with an NP-40 extract of EL-4 cells as well as Rauscher virus and Rauscher virus gp71. The slope of the competition curve with the cell extract was slightly shallower than that with virus or purified viral gp71. The amount of competing antigen was calculated by comparing the amount of protein required to give 50% competition with a standard preparation of Rauscher gp71. Precautions should be taken about interpreting this data quantitatively. First, inasmuch as the curves are plotted on a logarithmic scale, slight variations in drawing the slope can affect the value substantially. Second, because the assay measures

RELATED

TO

VIRAL

gpil

551

presumably only a small number of the antigenic determinants present on a molecule, the amount of antigen expressed as nanograms of protein may be only used for relative comparisons. Figure IA shows that. a xenotropic virus BALB/2 competed in this assay, but the competition was not complete and the amount required for 50% competition was about 8 times the amount required for 50% competition with Rauscher virus. In a homologous competition radioimmunoassay, 5-10 ng of Rauscher virus gp71 gave 50% competition, whereas in the interspecies assay 30-40 ng were required. Figure 1B shows competition curves for samples at different steps in the purification scheme. LDS extraction was used as the first step in purification since it was found to selectively solubilize surface proteins from tumor cells at concentrations below 5 n&f. Figure 2 shows the release of surface-labeled glycoproteins from the EL-4 cells with 3 mM LDS. Intact cells were labeled by treatment with neuraminidase and galactose oxidase followed by reduction with [SKIsodium borohydride. Total labeled glycoproteins in an NP-40 extract are shown for comparison. A presumed glycoprotein of about 70,000 daltons was certainly not a major cellular glycoprotein. After treat,ment with this concentration of LDS, the EL-4 tumor cells were microscopically intact but were 90% permeable to trypan blue. At concentrations above 6 mnM, there was

FIG. 1. A, interspecies competition radioimmunoassay. The assay was performed as described under Materials and Methods using 5 ng ‘““I-Rauscher virus gp71 (2 X lo4 cpm/ng) and goat anti-feline leukemia virus serum cl:2400 final dilution). An NP-40 extract of EL-4 tumor cells was used and viruses were disrupt,ed with 0.54 NP-40 and by freezing and thawing. B, analysis by interspecies competition radioimmunoassay of samples at various stages in purification of the cell surface antigen. 0, lithium diiodosalycilate extract of EL-4, A, DEAEpurified fraction, 0, LcH-Sepharose-purified fraction.

552

McLELLAN

A

B

224-

168-

112-

56-

27-

FIG. 2. Fluorogram of glycoproteins separated by SDS-polyacrylamide gel eleetrophoresis. Surface glycoproteins of EL-4 cells were labeled with [“HIsodium borohydride as described under Materials and Methods. A, total surface glycoproteins of EL-4 tumor cells. The labeled cells were disrupted with NP-40, the sample was clarified by centrifugation at 15,000 R for 15 min and an aliquot (80,000 cpm) was electrophoresed. B, cells were extracted with 3 n& lithium diiodosalycilate at room temperature for 30 min; the cells were then separated by centrifugation at 2000 g and an aliquot of the supernatant (35,000 cpm) was electrophoresed. The molecular weight markers were

AND

IHLE

cell lysis. The chromatographic profile of the extract on DEAE-cellulose is shown in Fig. 3. Use of gradient elution did not improve the purification. Affinity chromatography on lentil lectin-Sepharose and elution with LYmethyl mannoside gave substantial purifitkation. The recovery from the affinity column varied between 40-80%. If crude extracts were chromatographed on the affinity column, omitting the DEAE step, recovery and purification were poor. Table 2 summarizes the purification of a typical preparation starting with EL-4 tumor cells isolated from 30 mice. The overall purification in these steps varied from 250- to 600-fold. Greatest variation in yield was at the affinity chromatography step. The staining pattern with Coomassie blue of different fractions in the purification scheme separated by SDS-acrylamide gel electrophoresis is shown in Fig. 4. A doublet around 70-75,000 daltons and other very minor bands were found with the a-methyl mannoside elution. Rechromatography of the affinity column purified fraction on DEAE-cellulose gave a preparation displaying a single band of about 75,000 daltons by SDS-gel electrophoresis (Fig. 4F), but the recovery was poor. The fraction eluted from the affinity column with 1 M glycine (pH 3.0) showed one band at 75,000 daltons and a lesser band at 20,000 daltons (Fig. 4E). The low-molecular-weight protein leached from an affinity column developed with glycine in absence of added sample. It could be separated from the antigenically active band by gel filtration on Sephadex G150 (Fig. 4G). Serological characterization of the purified molecule. The purified cell surface gp75 was labeled with “‘1 and the reactivity with antisera to C-type viruses or viral glycoprotein was examined (Fig. 5). The most complete immunoprecipitation (45% of input count) was with goat anti-Rauscher gp71 or goat anti-Friend gp71 sera. Both chymotrypsinogen (27,ooO daltons), Rauscher MuLV gp71 (71,000 daltons), and glutamic dehydrogenase cross-linked with dimethylsuberimidate (multiples of 56,ooO daltons). The dried gel was exposed to BP 54 film (Kodak) for 3 days at -70”.

CELL

SURFACE

ANTIGEN

antisera are broadly reactive. There was also good reactivity to a goat antiserum to disrupted Moloney leukemia virus (40% precipitation), although there was no reactivity with an antiserum to purified Moloney virus gp71. Antisera against xenotropic virus (C57L virus) and feline leukemia virus precipitated some antigen, but there was practically no reactivity with antiserum against AKR gp71. In all cases, SDS-gel electrophoresis showed immune precipitates of about 70,000 daltons (data not shown). Either there is more than one species of about 70,000 daltons or the purified antigen shares some antigenic determinants with Rauscher, Moloney, Friend, C57L, and feline leukemia viruses. Competition radioimmune assays are mar? specific than immunoprecipitation in the type-specific serological characterization of an antigen. The purified cell surface antigen did not compete with Rauscher vi.:,-

Fir. 3. DEAE-cellulose chromatography. The extract was chromatographed and assayed as described under Materials and Methods. The solid line is the absorbance at 280 nm and the open circles are the amount of antigen determined by interspecies radioimmunocompetition expressed as micrograms of gp71.

PURIFICATION Protein (mg)

RELATED

TO

VIRAL

gp7l

rus gp71 or AKR virus gp71 in homologous assays. However, it was found to compete substantially but not completely in an assay xenotropic virus gp71 with “,‘I-BALB/B and antiC57L serum (Fig. 6A). The slope of the competition curve was shallower than with homologous virus, which could A

I3 c

1140 I35 :305 56. I 24.3 26.6 1.0

F

D E

G

-13 FIG. 4. SDS-polyacrylamide gel electrophoresis. Proteins separated on a 5-20%’ SDS polyacrylamide gel were stained with Coomassie blue. A, NP-40 extract of EL4 tumor cell (150 pg protein). B, lithium diiodosalycilate extract (200 pg protein). C, DEAE fraction (175 pg protein). D, fraction eluted from LcHSepharose column with u-methyl mannoside (10 Irg protein). E, fraction eluted from LcH-Sepharose coumn with I M glycine, pH 3.0 (I2 (~g protein). The molecular weight markers expressed in thousands were cytochrome c (MW, 13,000), Kauscher virus p.30 (MW, 30,000), ovalbumin (MW, 45,000), bovine serum albumin (MW, 68,000), Rauscher virus gp71 (MW, 71,000), and phosphorylase A (MW, 94,090). Lanes F and G are a separate gel of samples used for structural studies. A IO-16”l acrylamide gel was used to achieve maximum resolution in the 70-80.000-dalton region. F, (k-methyl mannoside fraction (lane D) rechromatographed on a DEAE column. G, Glycine-eluted fraction (lane El purified on a G-150 Scphadex column.

TABLE 2 OF CELL SURFACE ANTIGEN ~~ ~~ -.-.Interspecies

Wmg)

(14)

0.170 0.580 0.109 2.5

0.062 0.1 11 52.7

193.8 78.3 Xl 140

gpil (‘: rec‘oI’-

~~ Total LDS extract I LDS extract 2 I)EAE pool LcH Sepharose Wash I Wash 2 o-methyl mannoside

553

~-

(purification)

T!?Y’ ~~~ ~_ 100 40.4 17.1 7’2

____ 1 3.4 .ti4 14.7

1.5 2.9 ‘54.6

‘28

310

_

554

McLELLAN

DILUTION

FIG. 5. Titration

of various antisera with surface glycoprotein. An iodinated sample (22,000 cpm) and a suitable amount. of carrier IgG were added to dilutions of antisera and, after 3 hr incubation at 37”, a predetermined amount of second antiserum was added and the samples were incubated at 4” overnight and washed and counted as described under Materials and Methods. 0, goat anti-Rauscher virus gp71; a, goat anti-Friend virus gp71; A, goat antiC57L virus IgG, 0, goat anti-feline virus serum; 0, rabbit anti-AKR

tzP71.

be interpreted as nonidentity but cross-reaction of the antigens. In this assay, both classesof xenotropic viruses compete fairly comparably (e.g., BALB/B virus and NZB virus). EL-4 gp75 also competed in an assay with “‘1-Maloney virus gp71 and anti-hloloney virus serum (Figure 6B), although it did not, compete in an homologous assay using purified Moloney gp71 and rabbit anti-Moloney gp71 serum. The former antiserum has subsequently been found to be quite broadly reactive. It may be recognizing group-specific determinants, and is definitely recognizing interspecies antigenic determinants. Competition in an interspeties assay with Rauscher virus gp71 and anti-feline virus serum was shown in Fig. 1B. The results suggest that the molecule isolated form the cell surface of the EL-4 tumor cell is not serologically identical with any virally coded gp71 thus far examined, but that it is a cross-reactive protein, sharing antigenic determinants in common with C-type viruses and murine C-type viruses. It is most closely related to murine xenotropic gp71. The incomplete precipitation

AND

IHLE

FIG. 6. Competition radioimmunoassays. A, the assay was performed using 4 ng BALB/c xenotropic gp71 labeled with ““I and goat antiC57L virus IgG (1:800 final dilution). Competing proteins are BALB/c virus and cell surface glycoprotein purified by LcHSepharose chromatography (LcH). B, the assay was performed with 5 ng ‘*“I-Maloney virus gp71 and goat anti-Moloney virus serum (I:5000 final dilution). Competing proteins are Moloney MuLV disrupted as in Fig. 1 and lentil lectin-purified cell surface antigen (LcH).

and inability to compete at levels of protein equivalent to amounts of purified viral proteins may be due to either the amount of cross-reactivity or loss of antigenicity in purification of the molecule. The material eluting from the lectin affinity column with glycine at pH 3.0, although it appeared extremely pure by SDS gels, had relatively poor antigenicity. We attribute this to exposure to low pH, inasmuch as further loss of antigenicity was found if the material was not neutralized immediately after elution from the column. Glycoprotein nature of gp75. Affinity of the cell surface antigen for LcH-Sepharose suggeststhat the molecule is a glycoprotein containing mannose residues. Further proof of the glycoprotein nature of the molecule was obtained by labeling the purified sample with [“HIsodium borohydride after treatment with neuraminidase and galactose oxidase, or by labeling of sialic acid residues by borotrit,ide reduction after mild periodate oxidation. A fluorogram of an SDS gel of labeled samples (daba not shown) demonstrated a band of approximately 75,000 daltons. The terminal and penultimate sugars of the glycopeptide are sialic acid and galactose, respectively, and are the same as those of Rauscher gp71 glycopeptide (McLellan and August, 1976). Stained and fixed gels of electrophoresed

CELL

SURFACE

ANTIGEN

samples could also be labeled with ““I-Con A, confirming the presence of mannose residues in the carbohydrate chain. The total amount of carbohydrate on the molecule was not determined, so further comparisons cannot be made with the oligosaccharide moiety of the envelope glycoprotein of murine oncornavirus. Tryptic peptide comparison of purified gp71 s and gp75s. A more exacting approach to studying relatedness of molecules than the serological approach is to examine the primary sequence of proteins. Tryptic peptide mapping of tyrosine containing peptides of iodinated proteins, although not as exacting as examination of total tryptic peptides by ninhydrin staining or amino acid sequencing, allows one to make good comparisons using very small amounts of protein. Analysis of iodinated tryptic peptides was performed on cell surface antigen and viral glycoproteins. The various gp70 molecules to be examined were iodinated under conditions that should only give monoiodinated tyrosines. The preparations were electrophoresed on SDS gels to ensure that minor impurities or degradation products would be eliminated and the 70,000dalton band cut out and analyzed as described in Materials and Methods. Figure 7 shows tryptic peptide maps of the major envelope glycoprotein of endogenous ecotropic (AKR) virus, two classes of xenotropic virus (BALB/c and NZB), and exogenous viruses (Rauscher MuLV and Moloney MuLV). Maps are also shown of EL4 surface antigen which eluted from lentil lectin-Sepharose with a-methyl mannoside and the fraction more strongly bound to the affinity column which eluted with 1 M glycine, pH 3.0. Figure 7, A and B illustrate the reproducibility of the method. Two different preparations of AKR gp71 were digested, chromatographed, and electrophoresed at different times using different reagents and solvents. The maps do not exactly superimpose, but the patterns are extremely similar. A cursory examination of the peptide maps of each of the viral glycoproteins as well as the two preparations of cell surface antigen shows that no two maps are identical and that there is considerable structural diversity between the an-

RELATED

TO

VIRAL

gp71

555

tigenically closely related molecules. AKR gp71, Rauscher gp71, and Moloney gp71 are quite dissimilar. The xenotropic NZB and BALB/c gp7ls have many peptide spots in common but are easily distinguishable. The peptide map of the antigen purified from the cell surface of EL-4 cells is not identical with that of any of the viral glycoproteins, although it appears to have some peptides in common with xenotropic viral gp71. Tryptic peptide maps of EL-4 gp75 and Moloney gp71 were very dissimilar, although there was suggestion of some serological similarity. The fractions eluting from the lentil lectin-Sepharose column with a-methyl mannoside and 1 M glycine, respectively, have many peptides in common but there are some differences in the tryptic peptides. In Fig. 7, G and H the variable intensity of the spots accentuates the differences, but when tryptic peptides of the two samples were mixed and fingerprints performed, there was overlap of most of the spots and only a few unique spots were found (data not shown). The results clearly demonstrate that cell surface glycoproteins partially related to viral glycoproteins can be purified from nonvirus-producing cell lines. GPTl is a major determinant in the host range and interference properties of C-type oncornaviruses (Hunsmann et al., 19’74). The biological role of cell surface expression of this glycoprotein is not well understood. Occurrence of viral glycoprotein on the cell surface may prevent superinfection by a virus of the same serological class (Hunsmann et al., 1974). Also, the expression of the product of viral genes on the surface of tumor cells may have immunological significance in the regulation of tumor growth. Many strains of mice develop a natural immune response to viral proteins, in particular viral gp71 (Chle et al., 1974, 1976a), and the possibility exists that the cell surface proteins are immunological targets. Several reports have suggested widespread expression of oncornavirus glycoproteins in the absence of complete virus expression on both tumor and normal cells (Kennel and Feldman, 1976; Lerner ef al.,

FE. 7. Tryptic peptide maps of I”’ I-labeled murine viral gp7ls and cell surface gp75s. Tryptic peptide analysis was carried out as described in Materials and Methods. Chromatography was in the horizontal direction from right to left and electrophoresis in the vertical direction (upward from + to -). A and B are maps of AKH gp71 performed on different sampIes and with different reagents; C, Maloney gp71; D, Rauscher gp51; E, BALB/c xenotropic gp71; F, NZB xenotropic gp71; G, EL-4 surface antigen eluting from LcH-Sepharose with n-methyl mannoside; H, EL-4 surface antigen eluting from LcH-Sepharose with I M glycine pH 3.0.

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1975; Strand et al., 1974). Immunofluorescence with broadly reactive antisera or an interspecies competition assay has been used in these studies, so it is conceivable that what was being demonstrated was expression of a host gene and not a viral gene. Oncornaviral-like glycoproteins on cell surfaces (e.g., Grx alloantigen) have been implicated in cell differentiation (Stockert et al., 1971); the antiserum used in these studies is complex and not well defined. The anti-NTD serum which defines Glx differentiation alloantigen can detect three distinct but related 70,000-dalton glycoproteins (Tung et al., 1975a, 1976). Most typing antisera for studying cell surface antigens in the past have been prepared by immunization of syngeneic or alloge>eic mice with transplanted tumor lines The viral population of the cell lines has not been well characterized and the complexity of the antisera has led to some confusion in the literature. A recent paper reviewing surface antigens of tumor lines points out these complexities and the need to develop more specific reagents (Aoki et al., 1977). Characterization of related proteins by serological means is not always adequately sensitive to detect structural differences. Even a highly type-specific antiserum may not be able to detect microheterogeneity in the protein structure. Proteins of the size of oncornaviral glycoproteins have undoubtedly many antigenic determinants, including type, group, and interspecies antigenic determinants (Strand and August, 1973). Individual antisera prepared against the same protein may be directed against different determinants, resulting in preparations displaying either highly type-specific or broadly reactive antibodies. Structural comparison by tryptic peptide fingerprinting of purified glycoproteins from oncornaviruses, cell surfaces, serum, or other sources is a more rigorous and exacting way to compare relatedness. The isolation and purification of cell surface viral-like glycoproteins for structural studies is a formidable task compared with isolation of viral glycoproteins, inasmuch as the cell surface cont,ains a multitude of proteins. Furthermore, if virus is budding

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from the cell, viral glycoprotein may be isolated as well as cell surface glycoprotein. The EL-4 cell was chosen for study because it had a reasonable amount of “cross-react ing” glycoprotein and by electronmicroscopy was not a detectable virus producer (Aoki et al., 1977). However, it may contain a genome for oncornavirus because xenotropic virus could be isolated by co-cultivation techniques (Aoki et al., 19771, although it could not be detected by infection of rabbit cornea1 cells with extracts. The use of lentil lectin affinity chromatography was an important factor in purification of the cross-reactive antigen. The use of immunoaffinity chromatography with antiRauscher virus gp71 as used by Kennel (1976) to purify ascites fluid gp71 and by Elder et al. (1977a) to purify various virion gp71 was unsatisfactory in our hands. The latter investigators used immunoaffinity fo enrich for various gp7Osand then iodinated the protein and immunoprecipitated the partially purified sample with an ant.iRauscher gp71 serum to obtain material for tryptic peptide mapping. Unfortunately, no data on the purity of the preparations were shown (Elder et al., 1977a), and the number of tryptic spots observed is maximal considering the number of tyrosines determined by amino acid analysis of Rauscher gp71 (Marquardt et al., 1977). The glycoprotein isolated from the surface of the EL-4 tumor cell was serologically related to Moloney gp71 and xenotropic gp71; however, tryptic peptide analysis showed considerable differences from these glycoproteins, although there are possibly peptides in common. The structural diversity between gp7ls may reflect recombinational events between ecotropic and xenotropic proviral genes or recombinational events between host genes and viral genes. This may be an important mechanism for generating unique tumor surface antigens. In a recent paper, it has been shown that recombination occurs readily between endogenous ecotropic and xenotropic viruses and the enu gene which codes for gp71 may be a genetic hot spot (Elder et al., 1977b). Four isolates of the MCF group of leukemia viruses had unique tryptic peptide maps for gp71, whereas the products of the gag gene,

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p30 and ~15, were highly conserved structurally. Examination of tryptic peptide maps of gp7Os of many murine type C viruses by Elder et al. (1977a) has shown a great deal of structural divergency. This has led to the thought that gp7Os are polymorphic products of a large multi-gene family of immunologically and structurally related proteins. It is possible that oncornavirus glycoproteins have evolved from cellular antigens or vice-versa, but the possibility exists that tissue gp70-like molecules are not linked to oncornavirus glycoproteins at all. Isolation of sufficient quantities of the many different proteins in a form pure enough to do exacting structural studies or sequencing studies will be a formidable task. ACKNOWLEDGMENTS This work was sponsored by the National Cancer Institute under Contract NOI-CO-75380 with Litton Bionetics, Inc. REFERENCES AOKI, T., HERBERMAN, R. B., HARTLEY, J. W., LIU, M., WALLING, M. J., and NUNN, M. (197i). Surface antigens on transplantable tumor cell lines producing mouse type C viruses. J. Nat. Cancer Inst. 58, 1069-1078. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acid in polyacrylamide gels. Eur. J. Biothem. 46,83-X8. BOYSE, E. A., OLD, L. J., and CHOURALINKOV. I. (1964). Cytotoxicity test for demonstration of mouse antibody. Methods Med. Res. 10, 37-47. DEI, VILLANO, B. C., and LERNEK, It. A. (1976). Relationship between the oncornavirus gene product gp70 and a major protein secretion of the mouse genital tract. Nature 259,497-499. ELDER, J. H., JENSEN, F. C., BRYANT, M. L., and LF.HNER, R. A. (1977a). Polymorphism of the major envelope glycoprotein (gp70) of murine C-type viruses: Virion associated and differentiation antigens encoded by a multi-gene family. Nature 267, 23-28. ELDER, J. H., GAUTSCH, J. W., JENSEN, F. C., LERNEK, R. A., HARTLEY, J. W., and ROWE, W. P. (1977b). Biochemical evidence that MCF murine leukemia viruses are envelope (env) gene recombinants. Proc. Nat. Acad. Sci. USA 74,4676-4680. GOREK, P. A. (1950). Studies in antibody response of mice to tumor inoculation. Br. J. Cancer 4,372-379. HAYMAN, M. J., and CKUMPTON, M. J. (1972). Isolation of glycoproteins from pig lymphocyte plasma membrane using Lens culrnaris phytohemagglutinin. Biochem. Biophys. Res. Commun. 47, 923-930.

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HINO, S., STEPHENSON, J. H., and AARONSON, S. A. (1976). Radioimmunoassays for the i’O,OO@molecular weight glycoproteins of endogenous type-C viruses: Viral antigen expression in normal mouse tissues and sera. J. Vi&. 18, 933-941. HUNSMANN, G., MOENNIG, V., PISTER, L., SEIFERT. E., and SCHAFER, W. (1974). Properties of mouse leukemia viruses. VIII. The major viral glycoprotein of Friend leukemia virus. Seroimmunological interfering and hemagglutinating capacities. Virology 62, 307-318. HUNTER, W. M. (1975). Radioimmunoassay. In “Handbook of Experimental Immunology” (D. M. WEIR, ed.), pp. 17.1-17.36, F. A. Davis Co., Philadelphia. IHLE:, J. N., HANNA, M. G., JR., HOBERSON, L. E., and KENNRY, F. T. (1974). Autogenous immunity to endogenous RNA tumor virus: Identification of an tibody reactivity to select viral antigens. J. Exp. Med. 139, 1568-1581. IHLE, J. N., DF.NNE:Y, T. P., and BOI~O~NESI, -fi. P. (1976a). Purification and serological characterization of the major envelope glycoprotein from AKR murine leukemia virus and its reactivity with autogenous immune sera from mice. J. Viral. 17, 727-736. IHI.F,, d. N., .JOSEPH, D. R., and PAZMINO. N. H. (1976b). Radiation leukemia in C57BL/6 mice. II. Lack of ecotropic virus expression in the majority of lymphomas. +/. E’s!). Med. 144, 1406-1423. IHLE, J. N., and LAZAK, B. (1977). Natural immunity in mice to the envelope glycoprotein of endogenous, ecotropic type-C viruses: Neutralization of virus infectivity. J. Viral. 21, 974-980. KENNEL, S. J. (1976). Purification of a glycoprotein from mouse ascites fluid by immunoaffinity chromatography which is related to the major glycoprotein of murine leukemia viruses. Immunological and structural comparison with purified viral glycoproteins. J. Biol. Chem. 251, 6197-6204. KENNEI., S. .J.. and FELDMAN, J. D. (1976). Distribution of viral glycoprotein gp69/71 on cell surfaces of producer and nonproducer cells. Cancer Res. 36, 200-208. LF,RNER, H. A., WILSON, C. B., DEL VILLANO, B. C., MCCONAHEY. P. J., and DIXON, F. J. (1975). Endogenous oncornaviral gene expression in adult and fetal mice: Quantitative, histologic and physiologic studies on the major viral glycoprotein, gp70. J. Exp. Med. 143, 151-166. LEVY. ,J. A. (1973). Xenotropic viruses: Murine leukemia viruses associated with NIH Swiss. NZB and other mouse strains. Science 182, 1151-1153. LOWRY. 0. H., ROS~BKOUGH, N. ,J.. FARK, A. L., and RANDALL, R. d. (1951). Protein measurement with the Folin phenol reagent. ,I. Biol. C:hem. 193, 265-275. MAIZEI+ J. V., JR. (19i 1). Polvacrylamide electrophoresis of viral proteins. In “Methods in Virolog.y” (K.

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Maramarosch and H. Koprowski, eds.), Vol. 5, pp. 179-246, Academic Press, Inc., New York. MARQUARDT, H., GILDEN, R. V., and OROSZLAN, S. (1977). Envelope glycoproteins of Rauscher murine leukemia virus: isolation and chemical characterization. Biochemistry 16, 710-717. MCCLINTOCK, P. R., IHLE, J. N., and JOSEPH, D. R. (1977). Expression of AKR murine leukemia virus gp71-like and BALB(x) gp71-like antigens in normal mouse tissues in the absence of overt virus expression. J. Exp. Med. 146, 422-434. MCLEI,LAN, W. L., and ALZUST, T. J. (1976). Analysis of the envelope of Rauscher murine oncornavirus: in r*itro labeling of glycopeptides. .I. Viral. 20, 627-636. MOE:NNIG, V., FRANK, H., HUNSMANN, G., SCHNEIDER, J., and SCHAFER, W. (1974). Properties of mouse leukemia viruses. VII. The major viral glycoprotein of Friend leukemia virus. Isolation and physicochemical properties. Virology 61, 100-111. OHATA, Y., IKEDA, H., STOCKERT, E., and BOYSF., E. A. (19i5). Relation of Grw antigen of thymocytes to envelope glycoprotein of murine leukemia virus. J. Exp. Med. 141, 188-197. OROSZLAN, S., SUMMERS, M. R., FOREMAN, C., and GILDEN, R. V. (1974). Murine type-C virus groupspecific antigens: Interstrain, immunochemical, biophysical, and amino acid sequence differences, -J. Viral. 14, 1559-1574. STOCKERT. E., OLD, L. *J., and BOYSE, E. A. (1971). The G(x system: a cell surface alloantigen associated with murine leukemia virus: Implications regarding chromosomal integration of the viral genome. cJ. Exp. Med. 133, 1334-1355. STOCK~RT. E., BOYSF., E. A., OHATA, Y., IKF,DA, H.,

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SARKAR, N. H., and HOFFMAN, H. A. (1975). New mutant and congenic mouse stocks expressing I he murine leukemia virus-associated thymocyte surface antigen GLx. J. Exp. Med. 142, 512-516. STRAND, M., and AUGUST, J. T. (1973). Structural proteins of oncogenic ribonucleic viruses. Interspec. II. A new interspecies antigen. J. Biol. Chem. 248, 5627-5633. STRAND, M., LILLY, F., and AUGUST, J. T. (1974). Genetic control of the expression of murine leukemia virus proteins in tissues of normal, young adult mice. Cold Spring Harbor Symp. Quark Biol. 39, 1117-1122. Tu~o, .I.-S., FLEISSNER, E., VIT~TTA, E. S., and BOYSE, E. A. (1975a). Expression of murine leukemia virus envelope glycoprotein gp69/71 on mouse thymocytes. Evidence for two structural variants distinguished by presence vs. absence of G,x antigen. J. Exp. Med. 142, 518-523. TUNG, J-S., VITETTA, E. S., FLEISSNEK, E., BOYSE, E. A. (1975b). Biochemical evidence linking the G,x thymocyte surface antigen to the gp69/71 envelope glycoprotein of murine leukemia virus. J. Exp. Med. 141, 198-205. Turi-c, .I.-!%. QHEN, F.-W., FLEISSNER, E., and BOYSE:, E. A. (1976). X-gp70 a third molecular species of the envelope glycoprotein gp70 of murine leukemia virus, expressed on mouse lymphoid cells. J. Exp. Med. 143,969-974. WANG, C.-S., and SMITH, R. L. (1975). Lowry determination of protein in the presence of Triton X-109. Anal. Biochem. 63, 414-417. WF.BER, K., and KUTER, D. J. (1971). Reversible denaturation of enzymes by sodiurn dodecyl sulfate. cl. Biol. Chem. 246, 4504-4509.